U.S. Pat. No. 5,318,899 describes N6-(aminoiminomethyl)-N2-(3-mercapto-1-oxopropyl)-L-lysylglycyl-L-α-aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic (1→6)-disulfide of the formula (1) as a therapeutic agent for the treatment of, and prevention of, platelet-associated ischemic disorders. It binds to the platelet receptor glycoprotein (GP) of human platelets and inhibits platelet aggregation. Platelet aggregation is mediated by GP complex on the surface of the platelet membrane. It exists on the surface of unstimulated platelets in an inactive form. When platelets are activated by adhesion and the physiological agonists, the GP also becomes activated such that it becomes a receptor for fibrinogen, von Willebrand Factor (vWF), and fibronectin. However, it is the binding of fibrinogen and/or vWF that is believed to be principally responsible for platelet aggregation and thrombus formation in vivo. This teaches that substances, which specifically inhibit the binding of fibrinogen or vWF to GP, inhibit platelet aggregation and could be candidates for inhibiting thrombus formation in vivo (Eric J. Topol, Tatiana V. Byzova, Edward F. Plow and The Lancet; Vol 353; Jan. 16, 1999; pg 227-231). This article describes the compound having platelet aggregation inhibition activity but does not teach the method to synthesize the molecule.
Antagonists of platelet glycoprotein IIb/IIIa have an approved role in reducing the extent of thrombotic complications leading to myocardial damage during percutaneous coronary interventions (PCI).
Compound of formula (1) is a disulphide looped cyclic heptapeptide containing six amino acids and one mercaptopropionyl(desamino cysteinyl) residue. The disulfide bridge is formed between the cysteine amide and the mercaptopropionyl moieties. It is known to be produced by solution-phase peptide synthesis and purified by preparative reverse phase liquid chromatography and lyoplilized (www.fda.gov/cder/foi/label/1998/207181bl.pdf).
In terms of peptide synthesis methodology, two major synthetic techniques dominate current practice. These are synthesis in solution (homogeneous phase) and synthesis on solid phase (heterogeneous phase). But solution phase route is more cumbersome as compared to the solid phase route as after each coupling the peptide formed has to be isolated, whereas in the solid phase synthesis, the excess reagents and by-products are washed off by simple filtration. In both, the desired peptide compound is prepared by the step-wise addition of amino acid moieties to a building peptide chain.
U.S. Pat. Nos. 5,958,732 and 5,318,899 claim about recombinant techniques to synthesize peptides like N6-(aminoiminomethyl)-N2-(3-mercapto-1-oxopropyl)-L-lysylglycyl-L-α-aspartyl-L-tryptophyl-L-prolyl-L-cysteinamide, cyclic(1→6)-disulfide of the formula (1). The peptide obtained by this recombinant process is modified by solution phase synthesis for conversion of lysine residue to homoarginine residue. These patent documents also claim solid phase synthesis using Boc chemistry and the subject matter of these patents is fundamentally different from the present invention.
As compared to Boc-chemistry, Fmoc-chemistry based synthesis utilizes a mild procedure and because of the base lability of Fmoc group, acid-labile side-chain protecting groups are employed providing orthogonal protection. The rationale for use of protecting groups is that the energy of breaking a bond of a protecting group is lower than any other group.
U.S. Pat. Nos. 5,686,566, U.S. Pat. No. 5,686,567, U.S. Pat. No. 5,686,569, U.S. Pat. No. 5,686,570 and U.S. Pat. No. 5,756,451 deal with different PAI's in their salt or other forms of the compound of formula (1) but do not teach the process for its preparation using Fmoc solid phase synthesis.
Likewise, U.S. Pat. No. 5,759,999, U.S. Pat. No. 5,786,333, U.S. Pat. No. 5,770,564, U.S. Pat. No. 5,807,825, U.S. Pat. No. 5,807,828, U.S. Pat. No. 5,843,897, U.S. Pat. No. 5,968,902. and U.S. Pat. No. 5,935,926 describe the method of treating platelet-associated disorders and the process for the preparation of peptide amide of formula (1) using boc chemistry.
U.S. Pat. No. 5,344,783 and U.S. Pat. No. 5,851,839 deal with methods for selecting and identifying Platelet Aggregation Inhibitors (PAI) and disclose boc chemistry for the preparation of peptide amide of formula (1).
U.S. Pat. No. 5,780,595 claims antibodies specific to PAI's and also discloses boc chemistry for the preparation of the peptide amide of formula (1).
The Fmoc route of synthesis of various other peptides is well-known in prior art and several documents are available for their preparation. However there is a definite need to develop a process for the preparation of compound of formula (1) which is economical, involves minimal steps and also eco-friendly.
As explained earlier, Fmoc-chemistry based synthesis utilises a mild procedure and because of the base lability of Fmoc group, acid-labile side-chain protecting groups are employed providing orthogonal protection. The protecting groups used in Fmoc chemistry are based on the tert-butyl moiety: tert-butyl ethers for Ser, Thr, tert-butyl esters for Asp, Glu and Boc for Lys, His. The trt and acm groups have been used for the protection of Cys. The guanidine group of Arg and Har is protected by Mtr, Pmc or Pbf. Most of the Fmoc-amino acids derivatives are commercially available. However, a problem exists in the art for the preparation of some amino acid analogs like peptides containing homoarginine as well as cyclic peptide compounds based on disulfide links, because separate operations are required before purifying the end product, which increases expense and may affect final product purity and yield. Fmoc-homoarginine residue if purchased commercially for use in the assembly of the chain becomes expensive. Alternatively in the peptide assembly, the Har unit is built by guanylation of the lysine residue at the α-NH2 which has been demonstrated to obtain vasopressin analogues for the evaluation of its biological activity (Lindeberg et al, Int. J. Peptide Protein Res. 10, 1977, 240-244).
CN1500805 discloses preparation of Eptifibatide comprising: eliminating Fmoc protection of Fmoc-Rink Amide AM resin to obtain H2N-Rink Amide AM resin; connecting various protective amino acids successively to obtain corresponding resin; eliminating Fmoc-protection radical and Kaiser test to detect reaction procedure; preparing S-triphenyl mercapto propionyl-N, N-ditert butyl oxycarbonyl-homoarginine with lysine; grafting S-triphenyl mercapto propionyl-N,N-ditert butyl oxycarbonyl-homoarginine; eliminating side chain protecting radical and resin to reduce into coarse product; and cyclization, oxidation, HPLC tracking purification to obtain pure product.
WO 03/093302 discloses the synthesis of the peptide of formula (1) using Fmoc-α-nitrogen protected Cα-carboxamide cysteine. It describes the attachment of the first amino acid, cysteine in the protected form to the solid support 4-methoxytrityl polystyrene resin through its thiol side chain, followed by removing the α-nitrogen protecting group and assembling the peptide on the said resin. However, the process uses the solid support-4-methoxytrityl polystyrene resin which is not a common commercial embodiment and also the Fmoc-α-nitrogen protected Cα-carboxamide cysteine is not commercially available. This enables the process having increased number of steps and also expensive with respect to the process of the present invention. The cleavage conditions utilize ethanedithiol, which makes the process highly toxic and non-environment friendly requiring the use of expensive scrubbers. The use of Fmoc-homoarginine residue in the assembly of the chain is mentioned, which if purchased commercially, also makes the process very expensive. Overall, the process claimed in this document is different from the process claimed in the present invention. In addition the process of WO 03/093302 is associated with certain limitations, which has been overcome by providing suitable modifications in the process steps of the present invention.
A considerable number of known, naturally occurring small and medium-sized cyclic peptides as well as some of their synthetic derivatives and analogs possessing desirable pharmacological properties have been synthesized. However, wider medical use is often hampered due to complexity of their synthesis and purification. Therefore, improved methods for making these compounds in simple, lesser steps and at lesser cost are desirable and it is the need of the industry and mankind.
The purity and yield of the peptide are important aspects of any route of synthesis. Yield, represented by the relative content of the pharmacologically active compound in the final product, should be as high as possible. Purity is represented by the degree of presence of pharmacologically active impurities, which though present in trace amounts only, may disturb or even render useless the beneficial action of the peptide when used as a therapeutic agent. In a pharmacological context both aspects have to be considered. As a rule, purification becomes increasingly difficult with larger peptide molecules. In homogeneous (solution) phase synthesis (which is the current method of choice for industrial production of larger amounts of peptides) repeated purification required between individual steps provides a purer product but low yield. Thus, improvements in yield and purification techniques at the terminal stages of synthesis are needed. The present invention is an industrially feasible solid phase synthesis and is a novel process to yield a high purity product with enhanced yield.
Mutulis, F et al. discloses the use of a solid support system comprising cotton for multiple peptide synthesis (Journal of Combinatorial Chemistry, 5(1), January/February 2003).
Prior art describes the use of HOBT and DIC for activation of amino acids, which leads to the formation of Benzotriazole ester. However, a major drawback in using this procedure is that the preparation of the OtBu ester itself takes about 20 min and also the reaction has to be carried out at 0° C. The step-wise introduction of Nα-protected amino acids in SPPS normally involves in situ carboxyl group activation of the incoming amino acid or the use of pre-formed activated amino acid derivatives. In recent years, aminium and phosphonium based derivatives (HBTU, TBTU, Py Boc. and HATU) have become the preferred tools for in situ carboxyl activation. They have been shown to give superior results in terms of both coupling efficiency and suppression of enantiomerization. (Fmoc Solid Phase Peptide Synthesis by Chan W. C. and White P. D., Oxford University Press, 2000, p. 41-74) Use of HBTU provides high yield and high purity. It saves time in the activation step with no cooling required. Double coupling is also not required for Mpr(Acm)-OH.
Most of the Fmoc-amino acids derivatives are commercially available. However, a problem exists in the art for the preparation of some amino acid analogs like peptides containing homoarginine as well as cyclic peptide compounds based on disulfide links, because separate operations are required before purifying the end product, which increases expense and may affect final product purity and yield. Fmoc-homoarginine residue if purchased commercially for use in the assembly of the peptide chain becomes very expensive. Alternatively the peptide assembly can be built using lysine followed by guanylation of the lysine residue at the α-NH2 (Lindeberg et al., Int. J. Peptide Protein Res. 10, 1977, 240-244).
Fmoc-Lys(Boc)-OH is recommended for the routine preparation of Lysine containing peptides. For carrying side-chain modification of the Lys residue on the solid support, derivatives such as Fmoc-Lys(Mtt)-OH, Fmoc-Lys(ivDde)-OH, Fmoc-Lys(Mmt)-OH, Fmoc-Lys(Dde)-OH can be used since their respective side-chain protecting groups can be removed selectively on the solid-phase. (Rohwedder, B., et al.; Tetrahedron Letters, 39(5), 5 Mar. 1998, pp 1175-78 & Chhabra, S. R., et al.; Tetrahedron Letters, 39(12), 19 March 1998, pp 1603-06).
Oxidative cyclization of protected or non-protected sulfhydryl groups with formation of disulfide structures is usually carried out as the final synthetic step, the reason being substantial thermal and chemical lability of the disulfide linkage. In few cases it is also carried out before cleavage of the peptide molecule from the solid support. The oxidation of open-chain peptides containing free and/or certain types of protected sulfhydryl groups with iodine in, e.g., methanol or acetic acid is further explained in the CRC Handbook of Neurohypophyseal Hormone Analogs, Vol. 1, Part I: Jost, K., et al. Eds., CRC Press, Boca Raton, Fla. 1987, p. 31. Iodine, however, is not without drawbacks as a cyclization agent. For instance, tryptophan moieties present in peptide substrates are at risk of being iodinated, making the balance between full conversion of starting materials and minimizing side reactions a delicate one, which, in turn, impacts product purity. Tam (Tam J. P. et al., 1990, J. Am. Chem. Soc., Vol. 113, p. 6657) has demonstrated that the use of 20-50% solutions of DMSO in a variety of buffer systems greatly promotes disulfide bond formation in comparison with other methods such as aerial oxidation. DMSO is also found to greatly reduce and in some instances, suppress completely, the aggregation and precipitation of peptides that occurred using other oxidative procedures. Thus, the yield and purity of the disulfide looped peptide oxidized by DMSO is much higher as compared to other known methods. In the present invention this aspect has been rightfully tackled by not opting for Iodine route for oxidative cyclization. Thus the process steps of deprotection followed by oxidation of guanylated peptide amide adopted in the present invention yields crude peptide amide comprising compound of formula (1) of enhanced purity and yield. Finally purification of the crude peptide result in enhanced yield of the final pure peptide.
Another complicating factor in known routes of synthesis is the possibility of interaction between the desired cyclic disulfide and inorganic sulfur compounds used for reducing excess iodine at the end of the reaction, such as sodium dithionite or sodium thiosulfate. Such reducing sulfur-containing compounds may interact with the disulfide linkage, which is sensitive to nucleophilic attack in general. As the process of the present invention has avoided use of iodine, the resulting products have high purity and related impurities are undetectable.
The process is accomplished in a few easy and simple steps. The use of solid phase synthesis makes the process simpler and the use of Fmoc-chemistry eliminates the use of harsh chemicals like HF, thereby not affecting the product stability. The process eliminates purification of the intermediates, thereby increasing the yield and reducing the cost. Replacement of thiols as scavengers in step (b) and (e) makes the process more environment friendly and economical by not having to use scrubbers for thiols.
The choice of process often dictates the stability of the therapeutic peptide. There has been a long awaited requirement for obtaining peptide of formula (1), which will circumvent the limitations associated with the processes of prior art. Therefore, an industrial process of peptide synthesis containing tryptophan, disulfide loops, ε-NH2 side chain, etc demands appropriate choice of protecting groups and reaction conditions to build up the peptide chain. This objective has been now successfully achieved by the Applicant developing a process described in entirety in the present application.
Glossary of terms used hereinAAAmino acidAcmAcetamidomethylACTActivatorADPAdenosine diphosphateAgOTfSilver trifluoromethane sulfonateArgArginineAspAspartic acidBoc/boctert-butyloxycarbonylCysCysteineDCMDichloromethaneDde1-(4,4-dimethyl-2,6-dioxoyclohexylidene)ethylivDde1-(4,4-dimethy1-2,6-dioxoyclohexylidene)-3-methyl butylDEPDeprotection reagentDMFDimethyl formamideDMSODimethyl sulphoxideDTTDithiothreitolEDTEthane dithiolFmoc9-fluorenylmethyloxycarbonylGluGlutamic acidGlyGlycineHARHomoarginineHBTU2-(1H-Benzotriazole-1-y1)-1,1,3,3-tetramethyluroniumhexafluorophosphateHFHydrogen fluorideHICHydrophobic Interaction ChromatographyHisHistidineIECIon Exchange ChromatographyLC-MSLiquid Chromatography-Mass SpectroscopyLysLysineMmt4-methoxytritylMprMercaptopropionic AcidMtr4-methoxy-2,3,6-trimethylbenzenesulfonylMtt4-methyltritylNMMN-methyl morpholineO-t-BuO-t-butylPbf2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonylPmc2,2,5,7,8-pentamethylchroman-6-sulfonylPPPPlatelet poor plasmaProProlinePRPPlatelet rich plasmaRP-HPLCReverse Phase High Performance Liquid Chromatography.RVReaction VesselSerSerineSOLVSolventSPSynthetic PeptideTEATriethylamineTFATrifluoroacetic acidThrThreonineTISTriisopropylsilaneTrpTryptophanTrtTrityl